Molugu, T. | Search bioRxiv

bioRxiv2026

An Atomistic Description of Heterotypic Lipid Exchange by Sec14-like Phosphatidylinositol Transfer Proteins

Lipid transfer proteins (LTPs) are core regulators of the membrane dynamics, lipid signaling and intracellular communication networks that connect every organelle in the eukaryotic cell. ATP-independent lipid exchange reactions are a hallmark activity of these proteins. These remarkable reactions are essential for the important biological functions of LTPs but how lipid exchange is executed is not at all understood. Herein, we focus on phosphatidylinositol transfer proteins (PITPs) of the highly conserved and highly expanded Sec14/CRAL-Trio-like protein superfamily that potentiate phosphatidylinositol-4-phosphate (PtdIns4P) signaling in eukaryotic cells. Using an integrated structural approach, we describe in atomistic detail the lipid exchange reaction of Sec14-like PITPs. The molecular concepts we identify not only yield insights into how these PITPs integrate metabolic activity with PtdIns4P signaling in cells but also provide a framework for interpreting the functional mechanisms of other LTPs of the Sec14/CRAL-Trio superfamily.

biophysics↗

bioRxiv2025

Molecular mechanism by which SARS-CoV-2 Orf9b suppresses the Tom70-Hsp90 interaction to evade innate immunity

The Tom70-Hsp90 interaction is critical for activating MAVS-mediated interferon (IFN) production. Upon RNA virus infection, cytosolic Hsp90 recruits key innate immune signaling proteins to MAVS on mitochondria through its interaction with Tom70. To evade this innate immune response, the SARS-CoV-2 protein Orf9b binds to Tom70, thereby disrupting the Tom70-Hsp90 interaction and suppressing IFN production. Despite its importance, the molecular mechanism underlying Orf9b-mediated inhibition of IFN signaling remains unclear. Here, using an integrative approach combining cryo-electron microscopy, 19F NMR spectroscopy, and isothermal titration calorimetry (ITC), we show that Orf9b inhibits Hsp90 binding to Tom70 through a bipartite mechanism. The helix and intrinsically disordered tail of Orf9b sterically block the access of two distinct structural units of Hsp90 to Tom70. We also find that Orf9b-mediated allosteric conformational changes in Tom70 do not contribute to the inhibition of the Hsp90 binding. Comprehensive structural, thermodynamic, and kinetic analyses further reveal that Orf9b primarily slows the association kinetics between Hsp90 and Tom70. Collectively, our results provide a high-resolution mechanistic framework for understanding Orf9b-mediated suppression of the host innate immune response.

biophysics↗